Journal of Protein Chemistry, Vol. 22, No.

3, April 2003 ( 2003)

Behavior of S1- and S2-Ovalbumin and S-Ovalbumin A1 in Urea Solution: Kinetics and Equilibria
Hugh A. McKenzie1,3 and Robert D. Frier2,4
Received December 19, 2002

The isolation of S-, S1-, and S2-ovalbumin from domestic hen egg R-ovalbumin and of two methods for S-ovalbumin A1 are described. The rst is by heat treatment of R-ovalbumin A1 and the second is of R-ovalbumin followed by fractionation on Sepharose. A kinetics and equilibrium study is made of their behavior in the presence of urea and compared with that of R-ovalbumins. As anticipated, the S-ovalbumins are much more resistant to urea than R-ovalbumins. Unlike the latter, S-ovalbumins equilibrium proles have a simpler sigmoidal shape. The unfolding of S1- and S2-ovalbumin is an order of magnitude slower than that of R-ovalbumin. Some possible structural differences between R- and S-ovalbumin forms and their signicance are discussed.
KEY WORDS: S-, S1-, and S2-ovalbumin; S-ovalbumin A1; isolation; urea; unfolding; kinetics; equilibria.

1. INTRODUCTION In their kinetics studies of the optical rotation change at 589 nm ([ ]589) for whole domestic hen egg ovalbumin in concentrated urea over the pH range 5.59.2, Simpson and Kauzmann (1953) found that there was a primary change that was not simple rst order followed by a secondary change. Furthermore, although the results were highly repeatable for a given preparation of ovalbumin, there was variation in the half times for different preparations of ovalbumin. The kinetics for [ ]589 change were conrmed generally by McKenzie et al. (1963) over the same pH range, but the changes in A287 and A293 were rst order over at least two-half times under some conditions (Glazer et al., 1963). Furthermore, the reactions were very rapid at pH 3.1. The denaturation was further complicated by the presence of association and aggregation reactions, especially at the higher pH values. Even
1

School of Chemistry, University College, University of New South Wales, Australian Defence Force Academy, Canberra, Australia ACT 2600. 2 John Curtin School of Medical Research, Australian National University, GPO Box 334, Canberra, Australia ACT 2601. 3 To whom correspondence should be addressed at School of Chemistry, University College, University of New South Wales, ADFA, Canberra, ACT 2600, Australia. E-mail: h.mckenzie@adfa.edu.au 4 Deceased December 8, 2001.

when attempts were made to minimize these reactions there was still some lack of reproducibility of different batches of ovalbumin. McKenzie considered that the urea studies should be extended to the effect of temperature on ovalbumin in the absence of urea (so-called heat denaturation). When he commenced supervision of M. B. Smiths thesis work on the effect of temperature on ovalbumin, the need to prepare ovalbumin from fresh hen eggs was stressed. Indeed, as discussed in McKenzie (2003), Smith (1964), and Smith and Back (1962, 1965), it is shown that, as eggwhite ages, with concomitant increase in pH, native ovalbumin (here called R-ovalbumin) undergoes a transformation to another form (S-ovalbumin). The two forms exhibit markedly different rates (S < R) in heat denaturation at pH 3. Indeed, this observation is still the basis for determining the proportion of S-ovalbumin in a given sample of ovalbumin. Subsequently, Donovan and Mapes (1976) suggested that on the basis of differential scanning calorimetric studies, R-ovalbumin may be converted to two different forms of S-ovalbumin (S1 and S2), depending on the length of time of storage or of heating in solution. In this article, methods are described for the isolation of the intermediate form (S1-ovalbumin) and the nal form (S2-ovalbumin). Equilibrium and kinetics studies by a variety of methods of the urea denaturation over a 215
0277-8033/03/0400-0215/0 2003 Plenum Publishing Corporation

216 range of pH and temperature of both forms are reported. The results are compared with our results for R-ovalbumin and R-ovalbumin A1, A2, and A3 (McKenzie and Frier, 2003), and the evasion of the precise structure of Sovalbumin is discussed.

McKenzie and Frier or unfolded states. Furthermore, it was justied to study the kinetics and equilibria of the S-proteins by comparable ORD and CD methods. The CD spectra of the native proteins and their signicance are considered further in section 4.

2. MATERIALS AND METHODS 2.1. General Materials and Methods were common to the Rovalbumin paper, as in McKenzie and Frier (2003).

3.2. Equilibrium Studies The unfolding equilibrium proles of S1- and S2ovalbumin as measured by ORD at various wavelengths are very similar (e.g., 350 nm; Fig. 1). They are displaced along the urea concentration scale relative to the R-ovalbumin proles (McKenzie and Frier, 2003). To obtain 50% of the maximum change, a urea concentration of 5 M was needed for R-ovalbumin, whereas S1and S2-ovalbumin require 6.2 M urea to reach a similar state of change. Unlike R-ovalbumin, the S1- and S2ovalbumin equilibrium proles have a simple sigmoidal shape, and the normalized curves (Fig. 2) are coincident at all wavelengths, except for a possible slight difference at 232 nm. This difference is, however, only slightly outside the range of experimental error.

2.2. S-Proteins In the preparation of S-ovalbumin, R-ovalbumin paste (prepared as described previously) was dialyzed against water, titrated with 0.1 M NaOH to pH 9.9, and heated at 55C for periods of 16 hr, 2 days, 3 days, and 6 days. The solution was then allowed to cool to room temperature (ca. 20C), and a pH 4.5 stopping solution was added. A small quantity of occulant precipitate was removed by centrifugation. The supernatant was dialyzed against water before use. The S-ovalbumin-A1 was prepared by two methods: heat treatment of R-ovalbumin A1, which was prepared as in our previous work (McKenzie and Frier, 2003); and heat treatment of R-ovalbumin, followed by fractionation on CM-Sepharose. The products appeared to be identical. The heat stability method of Smith and Back (1965) was used for the analysis of R- and S-ovalbumin preparations. Differential scanning calorimetry curves of one series of preparations were recorded by the late M. B. Smith. These data conrmed that the R-ovalbumin contained less than 5% (w/w) S-ovalbumin and that the conversions to S1- and S2-ovalbumin were essentially complete after 10 hr and 30 hr, respectively. There is probably some S2-ovalbumin present in our 16-hr S1-ovalbumin.

3. RESULTS 3.1. ORD and CD Spectra The ORD and CD spectra of native R- and Sovalbumins are very similar. The spectra of the S-protein in 9 M urea are comparable to that of R-ovalbumins under equivalent conditions (i.e., in 7 M urea). It is therefore likely that there are no major conformational differences between R- and S-ovalbumins, either in the native

Fig. 1. Comparison of equilibrium proles of normalized optical rotation at several wavelengths in the range 232400 nm for Sovalbumin in urea solution of various concentrations, apparent pH 3.7, I 0.1 mol dm3, 25C.

Behavior of S1- and S2-Ovalbumin and S-Ovalbumin A1 in Urea Solution These observations are consistent with a simple two-state mechanism. However, it will be shown later in this work that the kinetics results do not support such a mechanism.

217

Over a narrow range of pH (4.55.0), Eq. (1) is obeyed: Y = AeBt CeDt + E (1)

3.3. Kinetics Studies It was immediately obvious from the kinetics work that both S1- and S2-ovalbumin are much more resistant than R-ovalbumin to unfolding by urea. Under similar conditions of urea concentration, pH, and temperature, the unfolding of S1- and S2-ovalbumin was found to be an order of magnitude slower than that of R-ovalbumin. To obtain kinetics results over a sufcient number of half-times and to ensure that the reaction went to completion, a urea concentration of 9 M was used in most of this work. Because the R-ovalbumin results were obtained in 7 M urea, the rate constants are not directly comparable. The kinetics form of S1-ovalbumin reaction generally was found to be similar to that of R-ovalbumin described previously over the pH range studied.

where Y is the reaction coordinate, and A, B, C, D, and E are constants for particular reaction conditions. At higher pH, Eq. (2) is obeyed: Y = AeBt + CeDt + E (2)

The denaturation kinetics of S2-ovalbumin in urea obey Eq. (2) over the entire pH range studied. Under no conditions was Eq. (1) obeyed by S2-ovalbumin. The denaturation kinetics of S2-ovalbumin prepared by heat treatment for 2, 3, or 6 days are indistinguishable. The pH dependence of the rate constants B and D for S1- and S2-ovalbumin (Fig. 3) is similar to that of Rovalbumin. The reaction is rapid at low pH and decreases markedly up to pH 6. At higher pH, there is relatively little change in rate with increasing pH. The rate constants for S2-ovalbumin are the same as those of S1-ovalbumin

Fig. 2. Comparison of equilibrium proles of reduced mean residue rotation [m ] at 350 nm for ovalbumin S1 and S2 at various urea concentrations, pH 3.7, I 0.1 mol dm3, 25C.

Fig. 3. Time course of difference absorbance ( A) at 287 nm for S2ovalbumin (0.95 g dm3) in 9 M urea solution at various pH values in range 4.267.21, I 0.1 mol dm3, 25C.

218 up to pH 6, but are 40% lower than those of S1-ovalbumin at higher pH. However, this difference is small compared with that between S1- and R-ovalbumin. The kinetics form and rate constants of S2-ovalbumin A1 are very similar to those of whole S2-ovalbumin. A major difference between R-ovalbumin and S1and S2-ovalbumin is that the ratio of constants C/A ( 0.5 0.15) is independent of pH for S1- and S2-ovalbumin. The same ratio for R-ovalbumin was found to be denitely pH dependent. Figure 4 illustrates typical kinetics curves for S2ovalbumin unfolding over a range of pH. The temperature dependence of constants B and D (Figs. 5 and 6) and, hence, reaction rate were found to be of similar form to that of R-ovalbumin; a minimum occurring in both B and D near 25C. 4. DISCUSSION

McKenzie and Frier

The resistance of the ovalbumins to urea denaturation parallels their heat denaturation behavior. R-ovalbumin is heat denatured at lower temperature, is unfolded at lower urea concentration, and has the highest rate of unfolding in urea. S1-ovalbumin is more resistant to both heat and urea denaturation, and S2-ovalbumin even more so. However, the differences between S1- and S2-ovalbumin unfolding rates in urea are small compared with the differences between the R- and S-forms. It is possible that the mechanisms of S1-ovalbumin unfolding in urea solution are similar to those for Rovalbumin proposed by us (McKenzie and Frier, 2003); that is, at pH >5: NID where N is a native form, I is a stable intermediate and D is the unfolded form.

Fig. 4. pH dependence of rate constants B and D for various Sovalbumin preparations in 9 M urea solution, I 0.1 mol dm3, 25C.

Fig. 5. Temperature dependence of rate constant B for absorbance differences at 287, 293, and 233 nm for S-ovalbumin in 9 M urea, pH 6.0 (6.6), I 0.1 mol dm3, 25C.

Behavior of S1- and S2-Ovalbumin and S-Ovalbumin A1 in Urea Solution

219

Fig. 6. Temperature dependence of rate constant D for absorbance difference at 287, 293, and 233 nm for S-ovalbumin in 9 M urea, pH 6.0 (6.6), I 0.1 mol dm3, 25C.

At pH 4.55.0, there appears to be some refolding of the unfolded form to a new conformation (D ) NDD The N to D transition is too fast in this pH range to enable possible intermediates (I) to be identied and characterized, using the instrumentation available to us in this work (see, e.g., Kim and Baldwin, 1990).

The urea denaturation of S2-ovalbumin appears to follow the rst mechanism over the whole pH range. S2ovalbumin is apparently unable to refold to D . The ORD spectra of R- and S-ovalbumins have been found to be very similar in this work. The only other work on ORD spectra is the limited work of Cho (1969), who obtained similar results to ours. However, there is now a signicant volume of comparative work on CD spectra of R- and S-ovalbumins. This is summarized in Table 1. It is evident that the differences between R- and S-spectra are small. Furthermore, as only some authors measurements were done on pure R- and S-samples, it is not possible to make completely unequivocal comparisons. In their thoughtful review of ovalbumin, Huntington and Stein (2001) conclude on the basis of the comparative CD spectra of R- and S-ovalbumin by Huntington et al. (1995) that R-ovalbumin has a slightly higher helix content and lower antiparallel -sheet structure than S-ovalbumin (see also Kint and Tomimatsu, 1979). They conclude that S-ovalbumin is probably a more stable partially loop-inserted form of ovalbumin and propose an explanation for the lack of serpin-like inhibitory properties. Also, they calculate a secondary structure composition for R-ovalbumin that is consistent with the one derived from the X-ray crystal structure of Stein et al. (1991). Calculations we have made from our ORD and CD spectra do not give satisfactory agreement with the X-ray structure. This is almost certainly because of the fact that our CD/ORD equipment had inadequate penetration into the UV. Even our use of the Kronig-Kramers transform (Woody, 1996) did not solve this problem. Johnson (1990) has stressed the need to measure spectra down to at least 180 nm.

Table 1. Some CD Parameters for R- and S-Ovalbumin Protein R-Ovalbumin Worker McK /nm 222 213 200 192 [ ] 12,400 10,500 +1500 +18,500c Hu Pa McK [ ] E E E +18,500c S-Ovalbumin Worker Hu [ ] 10,510 9290 +4100 +18,480 Pa [ ] 11,000 10,500 ca. 2000 Ta [ ] 11,510 10,500 +5470 +17750 R-Ovalbumin/S-Ovalbumin Worker Ze [ ] 12,050 10,400 +1250 Ko [ ] 11,800 9600

[ ] [ ] 11,000 11,000 9780 10,000 +4100 ca. 2000 +19,800

Notes: [ ] Mean molar residue ellipticity in deg cm2 decimol1. Values given here of other authors results are interpolated from spectra in their papers. Where the values given in the original paper are in terms of l r , they have been converted to values of by the relationship [ ] 3300 ( l r ). The mean residue molecular mass has been taken as 111 for a chain length of 385 residues. R-ovalbumin/S-ovalbumin indicates measurements on samples containing varying amounts of R- and S-ovalbumin, the exact proportions usually unknown. Key to workers: McKenzie, this article; Ko, Cho (1969); Hu, Huntington et al. (1995); Pa, Paolinelli et al. (1997); Ta, Tatsumi, and Hirose (1997); Ze, Zemser et al. (1994). Superscript c denotes calculated value. E denotes equivalent to value for R within experimental error.

220 Despite considerable work, no differences between R-ovalbumin and S-ovalbumin have been found for a variety of characteristics (see, e.g., Burley and Vadehra, 1989). One of us (H.M.) was originally of the view that a covalent difference involving SH-SS exchange would be the basis of the R/S transformation. Work in his laboratory (McKenzie and Shaw, 1973; M. Huddy, H. A. McKenzie, and D. C. Shaw, unpublished data) and that of Thompson and colleagues (see Webster and Thompson, 1980) has failed to reveal any difference in the location of the cysteine and cystine residues in the two forms of the protein. Despite the careful nature of these studies, and the large reagent excess used, very rapid interchange in the unfolded state cannot be excluded completely (see Halliday et al., 1993, for -lactoglobulin). Despite these observations, Cho (1969) has found that N-ethylmaleimide seemed to prevent R-ovalbumin S-ovalbumin conversion. Another possible covalent difference that is obvious is one of deamidation in the R-ovalbumin S-ovalbumin conversion. It has been examined by Kato et al. (1986), who have suggested that deamidation of asparagine or glutamine occurs during the conversion. Further work needs to be done on this possibility. It should be noted that variable results have been obtained for the number of titratable groups, some workers obtaining no difference, but Nakamura et al. (1981) nding not only that is the S-ovalbumin form hydrodynamically more compact but that it has a greater number of titratable groups exposed to the surface. Stein et al. (1991) noted in their X-ray crystallographic studies that R-ovalbumin binds a metal ion [e.g., calcium-(II)]. However, in their study of metal ion binding properties, Goux and Venkatasubramanian (1986) found that manganese-(II), for example, was bound to all forms of R-ovalbumin and S-ovalbumin they studied. Thus, in our opinion, the difference in structure causing the stability differences has not been elucidated unequivocally. Although ovalbumin has not had any biological function assigned to it, its evolution as a member of the serpin group of proteins, albeit without protease inhibitory activity, makes it important that the structural changes be elucidated.

McKenzie and Frier sity, where this work was initiated. Geoffrey Malcolm gave considerable assistance and direction in the treating of the ORD and CD data in structure prediction, and so forth. He also provided H.M. with much valuable discussion. Margaret Huddy and Denis Shaw gave assistance and advice, especially with respect to cystine/ cysteine analysis.

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ACKNOWLEDGMENTS Grateful acknowledgment is due to the John Curtin School of Medical Research, Australian National Univer-